1. 毒物分析 免疫檢驗 賴滄海教授 03-04-2009

2. Characteristics of the Screening and Confirmation Assays  Screening Assays Easy to Perform (automation) Low cost Sensitivity Precision Accuracy  Confirmation Assays Accuracy Precision Sensitivity Low Cost Easy to Perform

3. 濫用藥物免疫檢驗試劑之原理 ( 一 ) 1. Homogenous Immunoassay 1)Enzyme immunoassay Enzyme Multiplied Immunoassay Technique (Syva EMIT), DRI. Cloned Enzyme Donor Immuno Assay (CEDIA). 2)Fluorescence immunoassay Fluorescence Polarization Immunoassay (FPIA). Abbot TDx. 3)Microparticle agglutination immunoassay Roche Abuscreen Online

4. 2. Heterogeneous Immunoassay 1)Radio immunoassay (Roche, DPC) 2)Enzyme Linked Immunoassay (DCB) 濫用藥物免疫檢驗試劑之原理 ( 二 )

5. EMIT 的原理 G6PD - ＊ (Full Enzyme activity) + G6PD- ＊ :Ab (↓Enzyme activity) + ＊ :Ab G6P + NAD + 6-phosphogluconate + NADH (↑A 340 ) G6PD - ＊ + ＊ in sample G6PD - ＊ + Ab → G6PD - ＊： Ab (↓Enzyme activity)

7. Schematic Illustration of CEDIA ED ＊ : Ab + EA No enzyme activity Active enzyme –– ＊–– ＊ EA + ED ＊ + Ab →Ab : ＊ + (EA-ED ＊ ) 4

8. Fluorescence Polarization Immunoassay (FPIA) Ab ： Ag + Ab ： Ag - Fl + Ag - Fl (Decreased Polarization) Ag Ab + Ag - Fl → Ab ： Ag -Fl (Polarized Light)

9. Schematic Illustration of RIA DRUG ＊ :Ab1 DRUG :Ab1 DRUG ＊ DRUG DRUG ＊ :Ab1 :Ab2 DRUG :Ab1 :Ab2 Ab2 + + Ab1 DRUG ＊ DRUG (ppt)

10. Schematic illustration of DCB EIA No enzyme activity wash +＊+＊ Plate -Ab + ＊ -Enz wash Enzyme Activity + substrate -＊-＊

11. Amphetamine analogs

12. Percent cross reactivity of amphetamine analogs with various immunoassays (1) Reagents Analyte ug/mL Cross reactivity (%) Beckman (DRI) Cobas I400 Syva Emit p Syva Emit II CEDIA (microgenics) AxSYM (Abbot) D-Amphetamine1151.488.8202.797.9 135.5 BDB1020.64.43.94.894.5137.5 4-Bromo-2,5- dimethoxyphenethylamine 100.0 1.30.02.80.0 Bupropion100.00.80.00.313.90.1 Cathinone100.00.1 0.02.40.0 Chlorpheniramine100.0 0.40.01.40.0 N-Desmethylselegiline101.72.64.121.549.92.1 ethylamphetamine108.82.95.220.4221.045.1 Fenfluramine109.23.26.75.0152.116.2 Ketamine100.0 0.10.00.40.0 MBDB1135.328.4127.812.5861.026.6

13. Percent cross reactivity of amphetamine analogs with various immunoassays (2) Reagents Analyte ug/mL Cross reactivity (%) Beckman (DRI) Cobas I400 Syva Emit p Syva Emit II CEDIA (microgenics) AxSYM (Abbot) MDA185.640.2122.268.986.1119.6 MDEA1012.43.48.615.2793.657.0 MDMA1101.828.1110.127.7978.0132.2 Mescaline100.30.0 0.1 Methadone100.20.00.70.00.90.0 Methaqualone100.10.05.60.00.80.1 Methcathinone100.90.04.40.02.30.0 Methylephedrine100.90.01.50.01.10.1 Methylphenidate100.10.00.10.01.00.0 Methylpseudoephedrine100.10.01.30.01.60.0 Norketamine100.10.00.70.02.40.0

14. Percent cross reactivity of amphetamine analogs with various immunoassays (3) Reagents Analyte ug/mL Cross reactivity (%) Beckman (DRI) Cobas I400 Syva Emit p Syva Emit II CEDIA (microgenics) AxSYM (Abbot) Norpseudoephedrine (cathine)107.41.22.71.43.00.0 Pheniramine100.10.01.80.01.40.0 Phentermine104.00.085.517.851.613.8 Phenopropanolamine (Norephedrine, PPA) 100.30.62.70.02.50.0 PMA (p-methoxyamphetamine) 1027.770.659.612.185.318.1 PMMA (p-methoxymethamphetamine) 149.325.452.011.0936.8146.1 Propoxyphene100.0 6.11.51.20.0 Pseudoephedrine100.70.01.50.01.40.0 Selegiline101.20.01.90.01.40.1

15. Percent cross reactivity of amphetamine analogs with various immunoassays (4) Reagents Analyte ug/mL Cross reactivity (%) Beckman (DRI) Cobas I400 Syva Emit p Syva Emit II CEDIA (microgenics) AxSYM (Abbot) 2C-H (2,5-dimethoxy- phenethylamine) 100.90.02.80.32.40.0 2C-I (4-Iodo-2,5- Dimethoxyphenethylamine) 100.20.02.10.02.30.0 2C-T-2 (2,5-dimethyl-4- ethylthiophenethylamine) 100.30.01.90.02.00.1 2C-T-7 (2,5-dimethoxy-4- propylthiophenethylami ne) 100.10.02.40.02.40.0 3,4-dimethoxyphenethylamine100.0 6.40.71.60.1

16. Percent cross reactivity of amphetamine analogs with various immunoassays (5) Reagents Analyte ug/mL Cross reactivity (%) Beckman (DRI) Cobas I400 Syva Emit p Syva Emit II CEDIA (microgenics) AxSYM (Abbot) DOB (4-bromo-2,5- dimethoxyamphetamine) 100.10.03.50.10.01.1 Methamphetamine1168.831.9401.9186.3288.4147.7 HMA ( 4-hydroxy-3- methoxyamphetamine) 105.91.011.15.03.60.1 HHMA101.40.44.40.08.81.8 MTA (4- methylthioamphetamine) 103.016.483.812.896.265.9 PCP100.10.0 1.715.6 Tramadol100.30.0 1.878.2

17. Opiate

18. Apparent cross-reactivity (%) of various opiate immunoassays AnalyteRoche RIADPC RIASyva EMITAbbott FPIACEDIADRI O6-Acetylmorphine14.9015.792.881? Codeine1980.06330111125100 Hydrocodone24.50138.3100.55930 Hydromorphine9.90.769.2105.46630 Morphine84.783.586.390.0100 Morphine-3-Glucuronide52.40.945.364.29450 Oxycodone0.103.418.43.13 Oxymorphine0.10.011.210.81.90.6

19. Benzodiazepine

20. Cross Reactivity of Benzodiazepine Immunoassay (1) Benzodiazepines Cross Reactivity(%) DRISyva Emit d.a.u.Roche RIA CEDIA (microgenics) AxSYM (Abbot) Alprazolam18321916158309107 α -Hydroxy Alprazolam 91616109923989 Chlordiazepoxide2331455523 Clobazam52150779249 Clonazepam27393513435 7-Amino Clonazepam172196324 Diazepam22942190166294140 Flunitrazepam55110326353 7-Amino Flunitrazepam3353137840 N-Desalkyl Flunitrazepam3237216340 7-Hydroxy Flurazepam25013998216082

21. Cross Reactivity of Benzodiazepine Immunoassay (2) Benzodiazepines Cross Reactivity(%) DRISyva Emit d.a.u.Roche RIA CEDIA (microgenics) AxSYM (Abbot) N-Desalkyl Flurazepam7096917728985 Lometazepam42975916954 Lorazepam38365716740 Midazolam2038887312065 Nitrazepam901191896**68 7-Amino Nitrazepam3238406636 Nordiazepam1538134592**260102** 4 ’ -Hydroxy Nordiazepam 2455538624 Oxazepam106**86**689462 Prazepam811195510815076 Triazolam1158479823476 α -Hydroxy Triazolam 492959220556

22. Adulterants that Affects the Immunoassays 1. In Vivo Adulteration. 2. In Vitro Adulteration. (1) Detecting Adulteration. (2) EIA are more likely to be affected. (3) Substances used as adulterants. Alcohols, Ammonia, Ascorbic acid, Bleach, Blood, Detergent, Drano, Lemon Juice, Peroxide, Salt, Soap, Visine, Urinaid.

23. 水分吸收區 CTCT 反應區： 抗體 - 色素結合物 樣品吸收區 C ：品管線（陽性反應線） T ：藥物結合線（陰性反應線） 陽性 陰性 C CTCT ( 一條藍色 C 線） ( 二條藍色線條）

26. Table 3. Effects of Adulterants in Urine on Immunoassay Results Immunoassay AdulterantAmpBarbBzdCocTHCOpiPCP NaCl EEEEEEE Base Buffer EF Lime-A-Way or Liquid Plumber RFERF Vinegar ERKEK EFRKERKEK Bleach CFCEFCKRRK Drano EFCK RECK RECKEFCK R EFCK RK Detergent/Soap EC REFC RECEC REFC REC RFC R Visine (benzalkonium Cl) E FEK FECEECK REE Golden Seal CFEFCKR Urinaid (glutaraldehyde) E KRCEECE REFC RE RCEF KR Klear (KNO2) E RK E RE RK DETOX EFRK TEST CLEAN EFR E=EMIT, F=FPIA, C=CEDI, R=RIA, K=KIMS Underlined abbreviations indicate a false positive result. Abbreviations without underlines indicate a false negative result. Sone of data are from a 1998 personal communication from Sal Salamone, OraSure Technologies, Inc., Bethlehem, PA and Yale Caplan, Baltimore, MD.

27. Thanks for you attentions!